Modulation of in vivo 3-deoxyglucosone levels.

Fructoselysine 3-phosphate is synthesized in vivo by the recently discovered fructoseamine-3-kinase (F3K) from fructoselysine and ATP and decomposes to lysine, P(i) and 3-deoxyglucosone (3DG). This pathway appears to dominate 3DG production in vivo, making it possible to modulate 3DG levels by stimulating or inhibiting the reaction. Present inhibitors are non-reacting substrate analogues with relatively high K (i) values and can inhibit F3K sufficiently in vivo to reduce 3DG in diabetic rat plasma by approx. 50%. Stimulation of the F3K pathway by feeding glycated casein causes an increase of 10-20-fold in plasma levels of 3DG and 3-fold in kidney tubules. Consequences of this increase were studied in two systems: the Eker rat, a model of susceptible kidney tubules; and birth rates in two rat strains. In both cases substantial pathological effects were observed. In the Eker rats, an approx. 3-fold increase in kidney lesions was observed ( P <0.00001). In both Fischer 344 and Sprague-Dawley rats, birth rates were reduced by 56% ( P <0.0001) and 12% ( P <0.015) respectively. These results suggest that inhibition of F3K is a promising new therapeutic target for diabetic complications, as well as other 3DG-dependent pathologies.

DYN 12, a small molecule inhibitor of the enzyme amadorase, lowers plasma 3-deoxyglucosone levels in diabetic rats.

3-Deoxyglucosone (3DG) is a highly reactive alpha-dicarbonyl sugar and potent protein cross-linker that is important in the formation of advanced glycation end products (AGEs), which have been postulated to lead to the development of diabetic complications. (1) Reducing 3DG levels in diabetics is a potentially effective therapy to slow the development of diabetic complications. Standard biochemical methods were used to isolate, identify, and characterize the enzyme responsible for the production of 3DG, in order to develop an effective therapeutic agent against this target. We have purified and characterized Amadorase, a fructosamine-3-kinase, and demonstrated both in vitro and in vivo that it is responsible for the production of 3-deoxyglucosone (3DG). A small molecule inhibitor of Amadorase, DYN 12, significantly lowered plasma levels of 3DG in diabetic (by 46%, p = 0.0116) and normal (by 43%, p = 0.0024) rats. These data are the first indications that it is possible to significantly reduce 3DG production in diabetics and thus possibly reduce the development of diabetic complications.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts.

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline.

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.

Quantitation of 3-deoxyglucosone levels in human plasma.

3-Deoxyglucosone (3DG), a reactive dicarbonyl, is an important intermediate in the formation of advanced glycation end products (AGEs). The AGEs are particularly important in diabetes since they have been correlated with the development of diabetic complications. Consequently, measurements of 3DG are likely to provide valuable insights into the role of this metabolite in the etiology of diabetic complications. While several methods of 3DG quantitation in human plasma have been previously published, a significant discrepancy (over 30-fold) exists in the reported values. Knecht et al. (Arch. Biochem. Biophys. 294, 130-137, 1992) have reported the levels of plasma 3DG in normoglycemics to be 61 nM, using a GC/MS procedure. In contrast to this, Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993) reported 3DG levels to be 1800 nM in normoglycemics, using a totally independent GC/MS method. To resolve this disagreement and fill the need for a robust assay for this dicarbonyl, suitable for absolute quantitation, a GC/MS procedure was devised for its measurement. Plasma samples were deproteinized either by ultrafiltration or by addition of ethanol as described by Niwa et al. (Biochem. Biophys. Res. Commun. 196, 837-843, 1993). 3DG in the ultrafiltrate or the supernatant was conjugated with 2,3-diamino-naphthalene to produce a stable adduct which was then converted to a silyl ether and analyzed by GC/MS. The analyte was monitored by selected ion monitoring at an m/z of 295 and 306 and quantitated using an internal standard of [U-13C]3DG. Using this approach, 3DG levels in plasma deproteinized by ultrafiltration were found to be significantly elevated from 58.5 +/- 14 (SD) nM in normoglycemics to 98.5 +/- 34 (SD) nM in type I diabetics. When deproteinization of the plasma was carried out using ethanol, the levels of 3DG from normoglycemic plasma were similar to those reported by Niwa et al. (1710 +/- 750 (SD) nM). These results suggest that 3DG levels measured by ultrafiltration may represent the free circulating 3DG and those obtained by ethanol extraction may represent aform of 3DG bound to a macromolecule (presumbaly protein).

Metabolism of phosphonium choline by rat-2 fibroblasts: effects of mitogenic stimulation studied using 31P NMR spectroscopy.

Phospholipid turnover increases with both mitogenic stimulation and oncogenic transformation (1-9). Recent 31P nuclear magnetic resonance (NMR) spectroscopy studies of human tumors, animal tumor models and cell systems have reported elevated phosphomonoesters with growth and oncogenic transformation, as well as changes in these levels associated with treatment (10). In order to gain insights into the mechanisms underlying these changes, we used a phosphonium analog of choline and 31P NMR spectroscopy to study choline metabolism in quiescent and mitogenically stimulated Rat-2 fibroblasts. Cell growth status of these cells has a significant effect on choline metabolism. While overall uptake of the analog was similar in both quiescent and growing cells, distribution among metabolite pools differed. Quiescent cells accumulate label in the phosphodiester pool, with little or none in the phosphomonoester pool. On the other hand, mitogenic stimulation resulted in a significant fraction of the label in the phosphomonoester pool.

Identification of galactitol 2-phosphate and galactitol 3-phosphate in the lens of galactose-fed rats.

Production of unusual phosphorylated metabolites in the lens is one of several changes caused by hyperglycemia. Sorbitol 3-phosphate (Sor-3P) and fructose 3-phosphate (Fru-3P) are two such compounds identified in the diabetic lens, and galactitol 2-phosphate (Gal-2P) and galactitol 3-phosphate (Gal-3P) are identified here in the galactosemic lens. These new compounds are the first example of galactitol metabolism in mammalian tissue other than liver. Sor-3P and Fru-3P are also present in the galactosemic lens, apparently synthesized directly from their precursors, sorbitol and fructose, which are elevated in the lens due to increased flux of glucose through the aldose reductase (AR) pathway. The NADPH necessary to support this increased flux is derived from activation of the hexose monophosphate shunt (HMPS), which is clearly demonstrated by a large increase in the concentration of sedoheptulose 7-phosphate (Sed-7P), a HMPS-specific metabolite. Additionally, during 3 weeks of galactose feeding, there is a dramatic increase in lenticular concentrations of galactitol, sorbitol, galactose, and fructose and a sharp decrease in inositol. Glucose remains unchanged. A precipitous loss of both phosphorylated and nonphosphorylated metabolites occurs after 3 weeks, possibly due to lens rupture.

31P-nuclear magnetic resonance evidence of an activated hexose-monophosphate shunt in hyperglycemic rat lenses in vivo.

Using 31P-nuclear magnetic resonance spectroscopy, we have identified elevated concentrations of sedoheptulose-7-phosphate (S-7-P) in lenses from three animal models of hyperglycemia: streptozotocin-induced diabetic rats, galactose-fed rats, and xylose-fed rats. This observation provides a unique and independent confirmation of the activation of the hexose monophosphate shunt (HMPS) pathway in the hyperglycemic lens in vivo. While the elevation in concentration of S-7-P was very dramatic, the other HMPS metabolites in these tissues were below the threshold of detection, as expected for the HMPS pathway near equilibrium. In terms of nonenzymatic glycation, these results suggest that the only HMPS metabolite of importance in the hyperglycemic rat lens is S-7-P. Although in the diabetic lens its role appears to be relatively minor, in the galactosemic lens this compound may be an important contributor to the increased production of advanced glycosylation end products.

Metabolism of fructose-3-phosphate in the diabetic rat lens.

Fructose-3-phosphate and sorbitol-3-phosphate are produced in diabetic rat lenses by a 3-phosphokinase. While sorbitol-3-phosphate appears to be an inert polyol phosphate, fructose-3-phosphate is a potent cross-linking agent and a potential in vivo source of 3-deoxyglucosone. The objective of this study was to investigate the production and decomposition of fructose-3-phosphate in the diabetic rat lens. The results indicate that this metabolite achieves a steady-state concentration of almost 1 mumol/g wet weight within 2 weeks after the onset of diabetes. These steady-state levels appear to be a consequence of a balance between its production from fructose and its further decomposition to 3-deoxyglucosone. This conclusion is supported by results from disappearance of fructose-3-phosphate in insulin-treated diabetic rats and in vitro incubations of fructose-3-phosphate with amines where production of 3-deoxyglucosone was detected using a number of different methods including mass spectrometry. In agreement with these results, elevated concentrations of 3-deoxyglucosone along with its detoxification product, 3-deoxyfructose, were detected in the diabetic rat lenses. Other sugars and sugar phosphates which were detectable in the diabetic rat lenses were glucose, fructose, glucose-6-phosphate, fructose-6-phosphate, and sedoheptulose-7-phosphate. In conclusion, results from this study suggest that fructose-3-phosphate and 3-deoxyglucosone are likely to be important contributors to the process of nonenzymatic glycation in diabetic rat lenses.

Production of fructose and fructose-3-phosphate in maturing rat lenses.

PURPOSE: A large increase in glycation of crystallins between 1 and 8 months has been demonstrated in lenses obtained from aging rats. The objective of this study was to investigate if an age-associated increase in the levels of any of the phosphorylated and nonphosphorylated sugars in the aging rat lenses could be correlated with this increase. METHODS: Lenses were obtained from Sprague-Dawley rats ranging in age from 2 to 20 months. Trichloroacetic extracts of these tissues were analyzed by using 31P-NMR for sugar phosphates and high-pressure liquid chromatography equipped with an electrochemical detector for sugars and polyols. RESULTS: Although no elevation in the lenticular glucose levels was observed, an age-associated increase in the concentrations of polyol pathway-associated metabolites--sorbitol, fructose, sorbitol-3-phosphate, and fructose-3-phosphate--was detected. In contrast, no significant changes were observed in glycolytic or pentose shunt metabolites. CONCLUSION: Aging lenses accumulate increased concentrations of fructose and fructose-3-phosphate. Because fructose-3-phosphate is a potent glycating agent and a potential in vivo source of 3-deoxyglucosone, its accumulation in the lens, along with fructose, may be a contributing factor in the age-associated increase of nonenzymatic glycation in rat lenses.

Characterization of a phosphonium analog of choline as a probe in 31P NMR studies of phospholipid metabolism.

Tumors and transformed cells have been shown by 31P NMR to contain elevated concentrations of two phosphomonoesters, phosphorylcholine and phosphorylethanolamine, involved in phospholipid metabolism. In order to understand the biochemical basis for these phenomena new methods are needed to allow for analysis of the relevant metabolic pathways in intact cells. One such promising tool may be phosphonium-choline, a 31P NMR-visible analog of choline in which the trimethyl-ammonium group of choline has been replaced with a trimethyl-phosphonium moiety. As shown previously [Sim et al. Biochem. J. 154, 303 (1976)], this compound is non-toxic and readily metabolized by cultured cells into phospholipids. In this paper we describe in greater detail some of the chemical and NMR spectroscopic properties of this material. Most significantly we show here that the chemical shift of phosphonium-choline is sensitive to the phosphorylation state of the analog and that the phosphonium nucleus is NMR-visible even after its incorporation into phospholipid. The unique properties of this analog should make it possible to use high-field 31P NMR to follow the flux of phosphonium-choline through the Kennedy pathway in intact perfused cells cultures.

Effect of pH on the bioenergetics of perfused porcine lenses.

Extracellular and intracellular pH has been shown to be an important physiological variable in many biological systems. However, studies on the effect of pH on the metabolic status of the mammalian lens have been few. It has been shown previously that a change in perfusate pH has a significant effect on the lens weight, ion transport, opacification and membrane potential. In this study we show that the bioenergetic status of the perfused pig lens, as assessed by 31P-NMR spectroscopy, is critically dependent on the pH of the bathing medium. At pH's of 7.6 and above, these perfused lenses maintained their nucleotide triphosphates concentrations steady for up to 4 days. In contrast, lenses perfused with identical media at pH 7.0, rapidly lost their nucleotide triphosphates. Our findings suggest that the pH of the extracellular pH may be an important parameter in maintaining the functional competence of the lens.

Identification of a putative tumor marker in breast and colon cancer.

Application of a recently developed phospholipid saponification procedure to malignant colon or breast tissue produces an unidentified phosphodiester resonance in the 31P nuclear magnetic resonance spectrum which appears to correlate with malignancy. Glycerol phosphodiesters were prepared from a malignant breast tumor by a Folch extraction of the tissue followed by saponification of the resulting phospholipids. These compounds were separated by ion exchange chromatography and the compound responsible for this new resonance was isolated and identified as glycerol 2-phosphoglycerol by high resolution nuclear magnetic resonance spectroscopy. Confirmation of this structure was achieved by chemical synthesis of glycerol 2-phosphoglycerol. Attempts to isolate the phospholipid responsible for this resonance showed that it is not derived from a new class of phospholipids but is most likely an artifact of the new saponification procedure used. Glycerol 2-phosphoglycerol is formed via glycerol 2,3-(cyclic) phosphate derived from phosphatidylcholine followed by reaction with glycerol. Malignant tissue is more prone to produce this compound than is nonmalignant tissue and the differences between these tissue types may be a form of phosphatidylcholine that is present in higher concentrations in malignant tumor membranes than in those of normal tissue.

Detection of fructose-3-phosphokinase activity in intact mammalian lenses by 31P NMR spectroscopy.

Recently we have identified two novel phosphorylated metabolites in the lenses of diabetic rats as sorbitol 3-phosphate (Sor-3-P) and fructose 3-phosphate (Fru-3-P). The latter compound is of particular interest since it is a potent glycating agent, which could account, at least in part, for the increased protein cross-linking and cataract formation in the lens of the diabetic rat. In order to gain insight into the mechanism of formation of these compounds, 31P NMR spectra of rat, pig, and rabbit lenses, perfused with media supplemented with glucose, fructose, or sorbitol, were acquired. Perfusion with fructose-supplemented media resulted in the production of Fru-3-P in all three species. This compound was not produced upon perfusion with fructose-deficient media. The identification of the newly synthesized material as Fru-3-P was confirmed by spiking perchloric acid extracts of the perfused lenses with synthetic Fru-3-P. Our results provide strong evidence for the existence of a fructose-3-phosphokinase in mammalian lenses. If this enzyme is present in human lenses as well, it will reinforce the hypothesis that this enzyme and its product, Fru-3-P, may play a role in diabetic cataractogenesis and that lenticular fructose-3-phosphokinase may provide another therapeutic target in the prevention and alleviation of diabetic cataracts.

Human retinal pigment epithelial cells cultured in hyperglycemic media accumulate increased amounts of glycosaminoglycan precursors.

Confluent human retinal pigmented epithelial cells were cultured on microcarrier beads in the presence of 5.6 or 26 mmol/l glucose with or without the aldose-reductase inhibitor Sorbinil (200 microM) for 2 wk. At the end of the incubation period, perchloric acid extracts were prepared and analyzed by 31P nuclear magnetic resonance spectroscopy. As assessed by this method, the phosphorylated metabolites of cells incubated with 5.6 or 26 mmol/l glucose differed significantly in the concentrations of a number of uridine diphosphate (UDP)-conjugated monosaccharides, which were elevated two- to threefold in cells incubated in 26 mmol/l glucose over control samples. The affected metabolites were identified (through a series of spiking experiments) to be UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and UDP-glucuronic acid. Coincubation of the cells with Sorbinil 200 microM in the presence of 26 mmol/l glucose had no effect on this accumulation. Under normal circumstances, these molecules selectively and sequentially are incorporated into the polysaccharide chains of glycosaminoglycans (GAGs), whose presence and distribution in the basement membranes is affected adversely by diabetes mellitus. These data suggest that the availability of the monosaccharide precursor is not the rate-limiting step for GAG synthesis in the presence of pathologic glucose concentrations. Thus, the lost GAG content in the basement membranes of diabetic patients may be caused by changes elsewhere in the biosynthesis and/or catabolism of the polysaccharide-linked protein molecules.

Structural studies of a phosphocholine substituted beta-(1,3);(1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110.

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.

Fructose metabolism in the human erythrocyte. Phosphorylation to fructose 3-phosphate.

In human erythrocytes, the first step in the metabolism of fructose is generally thought to be phosphorylation to fructose 6-phosphate catalysed by hexokinase. In variance with this assumption, we show here that fructose in these cells is metabolized primarily to fructose 3-phosphate by a specific 3-phosphokinase. This process has an overall estimated Km of 30 mM with respect to extracellular fructose and an apparent Vmax. of 0.6 mumol/h per ml. At a fixed concentration of fructose in the medium, the accumulation of fructose 3-phosphate was linearly dependent on the duration of incubation up to 5 h and was not affected by glucose. Once accumulated, fructose 3-phosphate appears to be degraded and/or relatively slowly metabolized, decreasing by only approximately 30% after a 12 h incubation in a fructose-free medium.

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes.

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.

Identification of a naturally occurring methyl-ester of phosphate, methyl-phosphorylcholine (methyl-2-(N,N,N trimethylamino) ethyl phosphate), in the eggs of the sea urchin S. purpuratus.

A novel metabolite of choline, phosphorylcholine methyl ester, has been identified in the eggs of S. purpuratus wherein it is present at approximately 1 mM concentration. To the best of our knowledge, this is the first instance of a phosphoryl-methyl-ester to be observed in nature. The compound appears to be species specific, since it has not been observed in other species such as L. pictus and P. depressus. In S. purpuratus its distribution is confined to the ovary, eggs and embryos, and is absent from young animals following metamorphosis.

Approaches to isozyme-specific inhibitors. 17. Attachment of a selectivity-inducing substituent to a multisubstrate adduct. Implications for facilitated design of potent, isozyme-selective inhibitors.

The synthesis is described of a methyl-C5' adduct of L-methionine and beta,gamma-ATP bearing a 6-S-n-Bu group in place of the 6-NH2 group of the parent adduct. The latter is a potent multisubstrate inhibitor in a model system consisting of the M-2 and M-T isozymes of rat methionine adenosyltransferase. When attached to ATP, the 6-S-n-Bu group induces selectivity for M-T inhibition by elevating affinity for the ATP site of M-T but not of M-2. In the above adduct it exerted a similar effect, expressed by selectivity and increased inhibitory potency toward M-T. This affords a second illustration of the ability of this approach to generate, relatively readily, a potent inhibitor with moderate isozyme selectivity. An overview is given of extensive evidence from the present series of studies that moderate (ca. 10-fold) isozyme selectivity is often exhibited by substrate derivatives bearing a single short substituent at a variety of atoms. This, together with features of another feasible approach to isozyme-selective inhibitor design, suggests an approach that has potential to facilitate the design of potent inhibitors that are both isozyme-selective and selective for a given metabolic conversion. It comprises (1) evaluation of the above type of substrate derivatives as inhibitors of a chemotherapeutically significant set of isozymes (target and nontarget), (2) attempted derivation of a potent multisubstrate adduct inhibitor of the isozymes, (3) attachment to such an adduct of one or more selectivity-inducing substituents revealed in the first step, and, if desired, (4) systematic modification of substituents with a view to obtaining enhanced potency and/or isozyme-selectivity.